Tcp
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for ˜5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The TCP family of transcription factors is named after its first characterized members (teosinte-branched1 (TB1), cycloidea (CYC) and PCNA factor (PCF); Cubas P et al. (1999) Plant J 18(2): 215-22). In Arabidopsis thaliana, more than 20 members of the TCP family polypeptides have been identified, and classified based on sequence similarity in the TCP domain into Class I (also called Group I or PCF group) transcription factors that positively regulate gene expression, and Class II (also called Group II or CYC-TB1 group) transcription factors that negatively regulate proliferation. All TCP transcription factors are characterized by a non-canonical predicted basic-Helix-Loop-Helix (bHLH), that is required for both DNA binding and homo- and hetero-dimerization (see Cubas et al. above).
One Class I TCP polypeptide, AtTCP20 (also named PCF1 orthologue), binds to the promoter of cell cycle and ribosomal protein genes, as reported in Li et al. (2005) PNAS 102(36): 12978-83). International Patent Application WO0036124 provides a nucleic acid sequence encoding a Class I TCP polypeptide (named VBDBP) and the corresponding polypeptide sequence. Expression vectors and transgenic plants comprising the aforementioned VBDBP nucleic acid sequence are described. In International Patent Application WO2004031349, transgenic Arabidopsis thaliana plants overexpressing (using a 35CaMV promoter) a nucleic acid sequence encoding a Class I TCP polypeptide (named G1938) are characterized. Retarded plant growth rate and development are observed.
CAH3
Carbonic anhydrase catalyses the reversible reaction H2CO3⇄H2O+CO2. There are 3 classes of carbonic anhydrases (alpha, beta and gamma), phylogenetically unrelated but sharing some similarities at the active site. In plants, all three classes exist. Carbonic anhydrases are present in chloroplasts, mitochondria (mostly gamma class) and cytosol, and may represent up to 2% of total soluble proteins in leaves. Carbonic anhydrase is important for ensuring efficient photosynthesis by maintaining CO2 concentration in cells at a suitable level. It is known that at atmospheric O2 and CO2 pressure, ribulose bisphosphate carboxylase (Rubisco) works at 30% of its total capacity, hence there is interest in improving the CO2 uptake mechanism in plants. Carbonic anhydrase expression is co-regulated with the expression of Rubisco, and plants generally maintain a constant carbonic anhydrase versus Rubisco ratio. It is furthermore reported that carbonic anhydrase may also limit photorespiration by providing C-skeletons for nitrogen assimilation under certain conditions. In plants with a C3 type of photosynthesis, most of the carbonic anhydrase activity is localized to the stroma of the mesophyll chloroplasts, whereas in C4 plants, most of the carbonic anhydrase is found in the cytoplasm of mesophyll cells.
The idea of using carbonic anhydrase for increasing CO2 assimilation has been formulated many times. In WO9511979, it is postulated that transforming a monocotyledonous plant with a carbonic anhydrase from a monocotyledonous plant the ability of carbon dioxide fixation would be improved and would result in accelerated plant growth. Other documents disclose methods for mimicking a C4 type photosynthesis in C3 plants thereby improving the efficiency of photosynthesis (for example U.S. Pat. Nos. 6,610,913, 6,831,217 or US 20030233670). In these approaches, a C4-like pathway is introduced in C3 plants by introducing and expressing a combination of various enzyme activities (such as phosphoenolpyruvate carboxylase (PEPC) or pyruvate orthophosphate dikinase (PPDK)) from C4 plants to increase CO2 fixation; expression of these genes is under control of C4 regulatory sequences, typically their native promoters. Although predicted however, these attempts did not result yet in plants with increased yield.
Clavata
Leucine-rich repeat receptor-like kinases (LRR-RLKs) are polypeptides involved in two biological functions in plants, i.e., growth and development on one hand, and defense response on the other. LRR-RLKs are transmembrane polypeptides involved in signal transduction, with from N-terminus to C-terminus: (i) a signal peptide for ER subcellular targeting; (ii) an extracellular receptor domain to perceive signals; (iii) a transmembrane domain; and (iv) an intracellular cytoplasmic serine/threonine kinase domain that can phosphorylate downstream target proteins, be phosphorylated by itself (autophosphorylation) or by other kinases, or be dephosphorylated by phosphatases.
LRR-RLKs comprise the largest group within the plant receptor-like kinase (RLK) superfamily, and the Arabidopsis genome alone contains over 200 LRR-RLK genes. Members of this family have been categorized into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members (Shiu & Bleecker (2001) Proc Natl Aced Sc USA 98(19): 10763-10768). The subfamily LRR XI comprises one of the most studied LRR-RLK, Clavata1 (CLV1; Leyser et al., (2002) Development 116:397-403), involved in the control of shoot, inflorescence, and floral meristem size.
The shoot apical meristem can initiate organs and secondary meristems throughout the life of a plant. A few cells located in the central zone of the meristem act as pluripotent stem cells. They divide slowly, thereby displacing daughter cells outwards to the periphery where they eventually become incorporated into organ primordia and differentiate. The maintenance of a functional meristem requires coordination between the loss of stem cells from the meristem through differentiation and replacement of cells through division. In Arabidopsis, the Clavata (CLV1, CLV2, and CLV3) genes play a critical role in this process, by limiting the size of the stem cell pool in these meristems.
Clavata1 mutants have been identified in Arabidopsis (Leyser et al. see above; Clark et al., (1993) Development 119: 397-418; Diévart et al., (2003) Plant Cell 15: 1198-1211), in rice (Suzaki et al., (2004) Development 131: 5649-5657), and in corn (Bommert et al., (2004) Development 132: 1235-1245). All mutants present an enlargement of the aboveground meristems of all types (vegetative, inflorescence, floral) due to ectopic accumulation of stem cells, leading often to abnormal phyllotaxy, inflorescence fasciation and extra floral organs and whorls. This phenotypic severity varies between the different Arabidopsis mutants, the weaker alleles presenting only a small increase in stem cell number, whereas the strong alleles have more than 1000 fold more stem cells compared with the wild type (Dievart et al., (2004) supra).
The number of carpels formed per flower and the extent of growth of the ectopic whorls are sensitive indicators of clv1 mutant severity (Clarke et al., (1993) Development 119: 397-418). Two weak Arabidopsis mutants, clv1-6 and clv1-7, contain lesions after the transmembrane domain, leaving the possibility that the polypeptides these alleles encode are actually expressed and located to the plasma membrane (Clarke et al., (1993) supra).
Transgenic Arabidopsis plants expressing the nucleic acid sequence encoding the full length CLV1 polypeptide under the control of the ERECTA promoter (ER; for broad expression within the meristems and developing organ primordial) do not present a disrupted meristem (Clarke et al., (1993) supra). Granted U.S. Pat. No. 5,859,338 provides for an isolated nucleic acid sequence encoding a Clavata1 protein, and modified nucleic acid sequences encoding a modified Clavata1 protein, and describes expression vectors comprising the aforementioned isolated nucleic acid sequences, and plants and plant cells comprising the aforementioned isolated nucleic acid sequences.